Development of a CRISPR/SHERLOCK-Based Method for Rapid and Sensitive Detection of Selected Pospiviroids

Pospiviroids infect a wide range of plant species, and many pospiviroids can be transmitted to potato and tomato. Pospiviroids continue to be a major production constraint as well as of quarantine concern for the movement of germplasm, and are regulated in several countries/regions. The USDA APHIS issued a federal order requiring all imported tomato and pepper seeds be certified free of six pospiviroids of quarantine significance. The six pospiviroids of quarantine interest include CLVd, PCFVd, PSTVd, TASVd, TCDVd, TPMVd. Currently, those six viroids are detected by real-time RT-PCR. CRISPR/Cas-based genome editing has been increasingly used for virus detection in the past five years. We used a rapid Cas13-based Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) platform for pospiviroid detection, determined the limits of detection and specificity of CRISPR-Cas13a assays. This platform combines recombinase polymerase amplification (RPA) with CRISPR and CRISPR-associated (CRISPR-Cas) RNA-guided endoribonuclease that is rapid and does not require expensive equipment, and can be adapted for on-site detection.


Introduction
Viroids are single-stranded, circular RNAs molecules with relatively small genomes of about 239-401 nt that infect only plants.Unlike viruses, viroids do not code for any protein.So far, 33 different viroid species have been characterized and grouped into two families, Avsunviroidae and Pospiviroidae [1].The family Pospiviroidae was named from its type species, potato spindle tuber viroid (PSTVd).The Pospiviroidae contains five genera, Pospiviroid, Hostuviroid, Apscaviroid, Cocadviroid and Coleviroid [2].Viroids in the family Pospiviroidae replicate in the nucleus using host DNA-dependent RNA polymerase II and their multimers are cleaved by host enzymes; In contrast, Avsunviroidae viroids replication takes place in chloroplasts.The nuclear-encoded chloroplast RNA polymerase transcribes the circular (+) RNA to a linear (−) strand concatemer, and the multimers are processed by ribozyme-mediated self-cleavage [3][4][5][6].
Pospiviroids can infect a wide range of plant species.All pospiviroids except iresine viroid 1 and the related portulaca latent viroid can be transmitted to potato and tomato, with similar symptoms observed under controlled conditions [11,12].In addition to mechanical Viruses 2024, 16, 1079 2 of 13 transmission [13], several pospiviroids were reported to be transmitted via pollen and seeds [14].
Pospiviroids continue to be a production constraint as well as a quarantine reason for new germplasm arriving in several countries and regions including the United States.The USDA APHIS issued a federal order requiring all imported tomato and pepper seeds be certified free of six pospiviroids of quarantine significance, or produced in countries where these pospiviroids are not known to occur.The six pospiviroids of quarantine interest include CLVd, PCFVd, PSTVd, TASVd, TCDVd, TPMVd.
Currently, viroids are detected by real-time RT-PCR.CRISPR/Cas-based genome editing has been increasingly used for virus detection in the past five years [15][16][17][18][19][20][21][22][23][24].Different from Cas9, both Cas12 and Cas 13 proteins possess collateral or trans-cleavage activity.Cas12 is an endonuclease guided by crRNA to find the target DNA sequence, and then cuts both strands and the collateral DNA.Cas13 is an RNA-guided RNase which cleaves singlestranded and collateral RNAs.In both Cas12-based DNA endonuclease-targeted CRISPR trans reporter (DETECTR) [25] and Cas13-based Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) [26] systems, fluorescence signals can be generated via separation of the quencher from the reporter DNA/RNA by target recognition.In this work, we used a rapid SHERLOCK platform for pospiviroid detection.This platform combines recombinase polymerase amplification (RPA) with CRISPR and CRISPR-associated (CRISPR-Cas) RNA-guided endoribonuclease.It is highly specific in differentiating closely related pospiviroid pathogens, does not require expensive equipment, and can be adapted for on-site detection.To our knowledge, this is the first report of using SHERLOCK to detect pospiviroids of quarantine interest.

Plant RNA Extraction
Total RNA was extracted from healthy and viroid-infected host plant seed using the RNeasy Plant Kit (Qiagen GmbH, Hilden, Germany).Briefly, about 100 mg of tissues per sample was grounded into fine powder with mortar and pestle in liquid nitrogen.Buffer RLT (450 µL) and 4.5 µL β-mercaptoethanol were added to the tissue powder.The lysate was transferred to a QIAshredder spin column and centrifuged for 2 min at 13,000 rpm.The flow-through supernatant was then transferred to a new microcentrifuge tube, and mixed with half volume of ethanol (96-100%).An aliquot (650 µL) of mixture was added to an RNeasy Mini spin column and centrifuged for 15 s at ≥8000× g (≥10,000 rpm).Buffer RW1 of 700 µL was added to the RNeasy spin column and centrifuged for 15 s at ≥8000× g.RNeasy spin column was washed with 500 µL of Buffer RPE twice.RNase-free water of 30-50 µL was added to the spin column and centrifuged to elute RNA.Healthy potato 2.4.Recombinase Polymerase Amplification (RPA) RPA primers were designed based on genome sequences of the six pospiviroids, following the guidelines of 100-140 nt amplicon sizes, 54-67 • C primer melting temperatures, 30-35 nt primer lengths, and a T7 RNA polymerase promoter sequence (5 ′ -GAAATTAATACGACTCACTATAGGG-3 ′ , 25 nt) appended to the 5 ′ end of one primer to allow T7 transcription.RPA reactions were carried out using Twist Amp TM Basic Kit (TwistDx, Maidenhead, UK) following the manufacturer's protocol.The RPA reaction was set up as follows: 5.9 µL resuspended RPA solution, 0.5 µL RPA forward primer (10 µM), 0.5 µL RPA reverse primer (10 µM), 0.2 µL ProtoScript RT (100,000 U/mL), 1.4 µL Nuclease-free water, 1 µL RNA sample, and 0.5 µL MgOAc (280 mM).RPA reactions were performed at 39 • C for 20 min.

Comparative Sensitivity Analysis between Real-Time RT-PCR and CRISPR-Cas13
Based-Detection Serial dilutions of synthetic pospiviroids RNA transcripts were used to compare the sensitivity of CRISPR-Cas13 and real-time RT-PCR [27]-based detection.RNA concentrations and purity were measured with a NanoDrop One C Spectrophotometer (Thermo Fisher, Waltham, MA, USA).cDNA synthesis was performed using Bio-Rad iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA).Bio-Rad SYBR Green Supermix, CFX96 Real-Time System, and the CFX Manager software were used for the quantification of pospiviroids gene expression levels.

Detection of Pospiviroids with CRISPR-Cas13a
Although 37 • C was reported as the optimal reaction temperature, RPA reactions were performed at 39 • C for 20 min.as this temperature yielded the strongest amplification.The RPA amplicons were cloned into pGEM-T Easy vector and was confirmed by sequencing.The RPA products were used as the template for the Cas13a-based detection step.Green fluorescence was observed from PSTVd RNA transcript under UV light using PSTVd combination 1.There were no fluorescence signals with water and healthy-potato RNA controls (Figure 1).Similar results were obtained from the remaining five pospiviroids RNA transcripts using their specific combinations.The fluorescence density was also measured by fluorescence plate reader for quantification purposes.Fluorescence value was 5000-7000 with about 10ng viroid RNA transcript, while 400-600 was with healthy host RNA (negative control).Using this platform, we also tested two negative and three PSTVd-positive plant RNA samples previously confirmed of their infection status by real-time RT-PCR, and got consistent results for all the samples.
Viruses 2023, 15, x FOR PEER REVIEW 5 of 15 concentrations and purity were measured with a NanoDrop One C Spectrophotometer (Thermo Fisher, Waltham, MA USA).cDNA synthesis was performed using Bio-Rad iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA).Bio-Rad SYBR Green Supermix, CFX96 Real-Time System, and the CFX Manager software were used for the quantification of pospiviroids gene expression levels.

Detection of Pospiviroids with CRISPR-Cas13a
Although 37°C was reported as the optimal reaction temperature, RPA reactions were performed at 39°C for 20 min.as this temperature yielded the strongest amplification.The RPA amplicons were cloned into pGEM-T Easy vector and was confirmed by sequencing.The RPA products were used as the template for the Cas13a-based detection step.Green fluorescence was observed from PSTVd RNA transcript under UV light using PSTVd combination 1.There were no fluorescence signals with water and healthy -potato RNA controls (Figure 1).Similar results were obtained from the remaining five pospiviroids RNA transcripts using their specific combinations.The fluorescence density was also measured by fluorescence plate reader for quantification purposes.Fluorescence value was 5000-7000 with about 10ng viroid RNA transcript, while 400-600 was with healthy host RNA (negative control).Using this platform, we also tested two negative and three PSTVd-positive plant RNA samples previously confirmed of their infection status by real-time RT-PCR , and got consistent results for all the samples.

Comparative Sensitivity Analysis between Real-Time RT-PCR and CRISPR-Cas13a Based-Detection
Serial dilutions of synthetic RNA transcripts of pospiviroids were made to compare the sensitivity of CRISPR-Cas13 and real-time RT-PCR-based detection.Real-time RT-PCR primers for pospiviroids genes are listed in Table 3. Pospiviroid genomes are closely related, therefore, it is difficult to design real-time RT-PCR primers that are specific to individual pospivirod.The real-time RT-PCR sensitivity assay showed lower LOD than CRISPR-Cas13a for most pospiviroids, with copy number 5.32E+03 for PSTVd, 1.06E+06 for PCFVd, 9.48E+04 for CLVd, 6.43E+04 for TASVd, 4.95E+04 for TCDVd and 3.13E+04 for TPMVd (Figure 4).The specificity of real-time RT-PCR primers used was also tested (Table 4, Figure 5).PSTVd real-time RT-PCR primers detected not only PSTVd RNA, but also CLVd, TASVd, and TCDVd RNA templates.PCFVd real-time RT-PCR primers specifically detected only PCFVd RNA template.CLVd real-time RT-PCR primers, besides CLVd RNA also detected TASVd and TCDVd RNA templates.TASVd-specific real-time RT-PCR primers, besides TASVd RNA, also detected CLVd, TCDVd and TPMVd RNA templates.TCDVd-specific real-time RT-PCR primers, besides TCDVd RNA, also detected CLVd and TASVd RNA.TPMVd real-time RT-PCR primers, besides TPMVd, detected all other five pospiviroids RNA templates.The Cq value of all real-time RT-PCR primers was more than 39 when using healthy host RNA as the control.Among all real-time RT-PCR primers, those targeting PCFVd had the highest specificity and amplified only PCFVd, whereas TPMVd real-time RT-PCR primers can be used as a universal primer set to detect all the six viroids.Overall, neither real-time RT-PCR-nor CRISPR-Cas13a-based detection has high LODs or viroid specificity.primers was more than 39 when using healthy host RNA as the control.Among all realtime RT-PCR primers, those targeting PCFVd had the highest specificity and amplified only PCFVd, whereas TPMVd real-time RT-PCR primers can be used as a universal primer set to detect all the six viroids.Overall, neither real-time RT-PCR-nor CRISPR-Cas13a-based detection has high LODs or viroid specificity.

Discussion
Viroids are the smallest plant pathogens and consist of naked, circular single-stranded RNA and do not code for any proteins [28].Consequently, serodiagnostic techniques for viroid detection are not applicable except for recently reported polyclonal antibodies against PSTVd RNA [29].Traditional viroid detection methods such as biological indexing are used for the identification of causal agents [30].Nucleic acid-based techniques including RT-PCR, real-time RT-PCR, RT loop-mediated isothermal amplification (RT-LAMP), isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN), micro-and macro arrays, next-generation sequencing (NGS), and CRISPR-Cas12/13 are being used for detection.These methods offer faster, more sensitive, more reliable, and on-site testing options for detecting viroid infections [31].
There have been several reports of detection of plant virus/viroid using the CRISPR-Cas12/13 platform.Li et al. [32] developed a plasmonic CRISPR Cas12a assay to visually detect the colorimetric signal emitted by grapevine red-blotch viral infection in the vineyard.Jiao et al. [19] used a similar CRISPR/Cas12a-RT-RPA visual platform for detection of multiple apple-infecting viruses and the apple scar skin viroid (ASSVd).Coupled with either RT-RPA or LAMP, the CRISPR-Cas12a module can be used to detect plant RNA [33] and DNA [34] viruses, respectively.Furthermore, the CRISPR-Cas12a platform is sensitive enough for species-specific detection of similar viruses, such as differential diagnosis of tobamoviruses tomato mosaic virus and tomato brown rugose fruit virus [35].Marqués et al. [18] applied the CRISPR-Cas12a and CRISPR-Cas13a/d systems for indirect detection of viral DNA amplicons and direct diagnosis of viral RNAs, respectively.Among these reports, the CRISPR-Cas12a is much more popular with only one [18] involving CRISPR-Cas13a, which is the basis of SHERLOCK.Moreover, CRISPR/Cas-based plant viroid detection is reported only for ASSVd [19] with the sensitivity of ASSVd assay being significantly lower than those for other plant viruses when using the same CRISPR/Cas12abased platform.
Here, we used the SHERLOCK platform for pospiviroid detection (Figure 6), although with a lower detection sensitivity, it has the advantage of on-site testing compatibility.The short sequence length and high homology made it difficult to establish an ideal detection method, except for the high-cost and time-consuming direct sequencing approach.
Viruses 2023, 15, x FOR PEER REVIEW Among all combinations tested, PCFVd combination 1 was the only one that was specific in all the methods used.Although most of the RPA primer and crRNA combinations did not show high specificity when the viroid concentrations were high, their specificities increased with the reduction of non-target viroid concentrations.For example, PSTVd combination 1 was PSTVd-specific as long as TCDVd copy number did not reach at least 4.95E+10.CLVd combination 1 detected viroids other than CLVd only when the copy numbers of TASVd, TCDVd, or TPMVd reached E+11 level.Similarly, TASVd combination 1 was specific to TASVd detection with relatively low copy numbers of CLVd, TCDVd, and TPMVd.Insufficient (copy number thresholds vary from E+10 to E+11) PSTVd, TASVd, or TPMVd did not interfere with the specific targeting of TCDVd combination to TCDVd either.In addition to TPMVd, the TPMVd combination was able to detect E+11 level PSTVd and TCDVd.In contrast, the copy numbers required for anticipated viroid detection using these combinations ranged from E+2 to E+7.Considering that viroid copy numbers in plant hosts can rarely reach E+10 to E+11 levels, our SHERLOCK platform may have adequate specificity in real-world viroid detection.
Due to the different nature of real-time RT-PCR and CRISPR-Cas13a reactions, the SHERLOCK platform had lower sensitivity as compared to the more traditional real-time RT-PCR approach.Real-time RT-PCR assays need to be performed in the lab with realtime PCR machine, and the data (Cq value) can only be obtained from the equipment.In contrast, the CRISPR-Cas13a-based viroid detection system can be adapted to on-site application with no need of costly instruments, and the output is visible under portable UV light in the field, which are critical advantages over real-time RT-PCR.In the future, SHERLOCK viroid diagnosis platform needs further refinement to improve its sensitivity to a level comparable to that of real-time RT-PCR.In addition, expanding the LOD gaps between targeted and non-specific viroids would further increase the specificity of the platform.On the other hand, it's difficult to apply the current multiple viroid detection system (results from specificity assay) for diagnosis purpose due to the high LOD values required, whereas, narrowing viroid LOD gaps and reducing their absolute LOD values would facilitate detection of up to all six regulated viroids.Owing to their relatively high Among all combinations tested, PCFVd combination 1 was the only one that was specific in all the methods used.Although most of the RPA primer and crRNA combinations did not show high specificity when the viroid concentrations were high, their specificities increased with the reduction of non-target viroid concentrations.For example, PSTVd combination 1 was PSTVd-specific as long as TCDVd copy number did not reach at least 4.95E+10.CLVd combination 1 detected viroids other than CLVd only when the copy numbers of TASVd, TCDVd, or TPMVd reached E+11 level.Similarly, TASVd combination 1 was specific to TASVd detection with relatively low copy numbers of CLVd, TCDVd, and TPMVd.Insufficient (copy number thresholds vary from E+10 to E+11) PSTVd, TASVd, or TPMVd did not interfere with the specific targeting of TCDVd combination to TCDVd either.In addition to TPMVd, the TPMVd combination was able to detect E+11 level PSTVd and TCDVd.In contrast, the copy numbers required for anticipated viroid detection using these combinations ranged from E+2 to E+7.Considering that viroid copy numbers in plant hosts can rarely reach E+10 to E+11 levels, our SHERLOCK platform may have adequate specificity in real-world viroid detection.
Due to the different nature of real-time RT-PCR and CRISPR-Cas13a reactions, the SHERLOCK platform had lower sensitivity as compared to the more traditional real-time RT-PCR approach.Real-time RT-PCR assays need to be performed in the lab with real-time PCR machine, and the data (Cq value) can only be obtained from the equipment.In contrast, the CRISPR-Cas13a-based viroid detection system can be adapted to on-site application with no need of costly instruments, and the output is visible under portable UV light in the field, which are critical advantages over real-time RT-PCR.In the future, SHERLOCK viroid diagnosis platform needs further refinement to improve its sensitivity to a level comparable to that of real-time RT-PCR.In addition, expanding the LOD gaps between targeted and non-specific viroids would further increase the specificity of the platform.On the other hand, it's difficult to apply the current multiple viroid detection system (results from specificity assay) for diagnosis purpose due to the high LOD values required, whereas, narrowing viroid LOD gaps and reducing their absolute LOD values would facilitate detection of up to all six regulated viroids.Owing to their relatively high sensitivity and

Figure 1 .
Figure 1.Visualization of fluorescence resulting from potato spindle tuber viroid (PSTVd) detection.From left to right, PSTVd RNA, water control, and healthy host RNA samples.

Figure 1 .
Figure 1.Visualization of fluorescence resulting from potato spindle tuber viroid (PSTVd) detection.From left to right, PSTVd RNA, water control, and healthy host RNA samples.

15 Figure 2 .
Figure 2. Sensitivity analysis of monoplex CRISPR-Cas13a based detection of six pospiviroids (A-F).All experiments were repeated three times with similar results obtained.X-axis represents the copy number of serially diluted RNA transcript.Y-axis represents the fluorescence value.

Figure 2 .
Figure 2. Sensitivity analysis of monoplex CRISPR-Cas13a based detection of six pospiviroids (A-F).All experiments were repeated three times with similar results obtained.X-axis represents the copy number of serially diluted RNA transcript.Y-axis represents the fluorescence value.

Figure 4 .
Figure 4. Sensitivity analysis of real-time RT-PCR detection of six pospiviroids (A-F).All experiments were repeated three times with similar results obtained.X-axis represents the copy number of serially diluted RNA transcript.Y-axis represents the Cq value.

Figure 4 .
Figure 4. Sensitivity analysis of real-time RT-PCR detection of six pospiviroids (A-F).All experiments were repeated three times with similar results obtained.X-axis represents the copy number of serially diluted RNA transcript.Y-axis represents the Cq value.

Figure 5 .
Figure 5. Specificity analysis of real-time RT-PCR primers (A-F).The Cq value of all real-time RT-PCR primers were more than 39 when water was used as the control.All experiments were repeated three times with similar results obtained.X-axis represents the RNA transcript.Y-axis represents the Cq value.

Table 3 .
List of real-time RT-PCR primers designed and used for the detection of viroids.